Biomarker normalization

ABSTRACT

A fluid sample is measured with a tear film measuring system that includes a processing device that receives a sample chip comprising a sample region configured to contain an aliquot volume of sample fluid, the processing device configured to perform analyses of osmolarity and of one or more biomarkers within the sample fluid, wherein the analysis of biomarkers includes normalization of biomarker concentration values.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Non-Provisionalapplication Ser. No. 11/358,986 entitled “Tear Film Osmometer” filed onFeb. 21, 2006 (attorney docket no. 021935-000311 US), which is acontinuation application of U.S. Utility application Ser. No. 10/400,617entitled “Tear Film Osmometer” filed Mar. 25, 2003 (now U.S. Pat. No.7,017,394), which claims priority to U.S. Provisional Patent ApplicationSer. No. 60/401,432 entitled “Volume Independent Tear Film Osmometer”filed Aug. 6, 2002. This application claims the benefit of priorityunder 35 U.S.C. 119 to U.S. Provisional Application Ser. No. 60/912,129entitled “Biomarker Normalization” filed on Apr. 16, 2007 (attorneydocket no. 021935-001100US). Each of these applications is incorporatedherein in its entirety as if set forth in full.

BACKGROUND

1. Field of the Invention

The field of the invention relates generally to osmolarity measurementsand more particularly to systems and methods for calibrating tar filmosmolarity measuring devices.

2. Background Information

Tears fulfill an essential role in maintaining ocular surface integrity,protecting against microbial challenge, and preserving visual acuity.These functions, in turn, are critically dependent upon the compositionand stability of the tear film structure, which includes an underlyingmucin foundation, a middle aqueous component, and an overlying lipidlayer. Disruption, deficiency, or absence of the tear film can severelyimpact the eye. If unmanaged with artificial tear substitutes or tearfilm conservation therapy, these disorders can lead to intractabledesiccation of the corneal epithelium, ulceration and perforation of thecornea, an increased incidence of infections disease, and ultimatelypronounced visual impairment and blindness.

Keratoconjunctivitis sicca (KCS), or “dry eye”, is a condition in whichone or more of the tear film structure components listed above ispresent in insufficient volume or is otherwise out of balance with theother components. It is known that the (fluid tonicity or osmolarity oftears increases in patients with KCS. KCS is associated with conditionsthat affect the general health of the body, such as Sjogrcn's syndrome,aging, and androgen deficiency. Therefore, osmolarity of a tear film canbe a sensitive and specific indicator for the diagnosis of KCS and otherconditions.

The osmolarity of a sample fluid (e.g., a tear) can be determined by anex vivo technique called “freezing point depression,” in which solutesor ions in a solvent (i.e. water), cause a lowering of the fluidfreezing point from what it would be without the ions. In the freezingpoint depression analysis the freezing point of the ionized sample fluidis found by detecting the temperature at which a quantity of the sample(typically on the order of about several milliliters) first begins tofreeze in a container (e.g., a tube). To measure the freezing point, avolume of the sample fluid is collected into a container, such as atube. Next, a temperature probe is immersed in the sample fluid, and thecontainer is brought into contact with a freezing bath or Peltiercooling device. The sample is continuously stirred so as to achieve asupercooled liquid state below its freezing point. Upon mechanicalinduction, the sample solidifies, rising to its freezing point due tothe thermodynamic heat of fusion. The deviation from the sample freezingpoint from 0° C. is proportional to the solute level in the samplefluid. This type of measuring device is sometimes referred to as anosmometer.

Presently, freezing point depression measurements are made ex vivo byremoving tear samples from the eye using a micropipette or capillarytube and measuring the depression of the freezing point that resultsfrom heightened osmolarity. However, these ex vivo measurements areoften plagued by many difficulties. For example, to perform freezingpoint depression analysis of the tear sample, a relatively large volumemust be collected, typically on the order of 20 microliters (μL) of atear film. Because no more than about 10 to 100 nanoliters (nL) of tearsample can be obtained at any one time from a KCS patient, thecollection of sufficient amounts of fluid for conventional ex vivotechniques requires a physician to induce reflex tearing in the patient.Reflex tearing is caused by a sharp or prolonged irritation to theocular surface, akin to when a large piece of dirt becomes lodged inone's eye. Reflex tears are more dilute, i.e. have fewer solute ionsthan the tears that am normally found on the eye. Any dilution of thetear film invalidates the diagnostic ability of an osmolarity test fordry eye, and therefore make currently available ex vivo methodsprohibitive in a clinical setting.

A similar ex vivo technique is vapor pressure osmometry, where a small,circular piece of filter paper is lodged underneath a patient's eyeliduntil sufficient fluid is absorbed. The filter paper disc is placed intoa sealed chamber, whereupon a cooled temperature sensor measures thecondensation of vapor on its surface. Eventually the temperature sensoris raised to the dew point of the sample. The reduction in dew pointproportional to water is then converted into osmolarity. Because of theinduction of reflex tearing and the large volume requirements forexisting vapor pressure osmometers, they are currently impractical fordetermination of dry eye.

The Clifton Nanoliter Osmometer (available from Clifton TechnicalPhysics of Hartford, N.Y., USA) has been used extensively in laboratorysettings to quantify the solute concentrations of KCS patients, but themachine requires a significant amount of training to operate. Itgenerally requires hour-long calibrations and a skilled technician inorder to generate acceptable data. The Clifton Nanoliter Osmometer isalso bulky and relatively expensive. These characteristics seriouslydetract from it use as a clinical osmometer.

In contrast to ex vivo techniques that measure osmolarity of tearsamples removed from the ocular surface, an in vivo technique thatattempted to measure osmolarity directly on the ocular surface used apair flexible pair of electrodes that were placed directly underneaththe eyelid of the patient. The electrodes were then plugged into an LCRmeter to determine the conductivity of the fluid surrounding them. Whileit has long been known that conductivity is directly related to theionic concentration, and hence osmolarity of solutions, placing thesensor under the eyelid for half a minute likely induced reflex tearing.Furthermore, these electrodes were difficult to manufacture and posedincreased health risks to the patient as compared to simply collectingtears with a capillary.

It should be apparent from the discussion above that current osmolaritymeasurement techniques are unavailable in a clinical setting and can'tattain the volumes necessary for dry eye patients. Thus, there is a needfor an improved, clinically feasible, nanoliter-scale osmolaritymeasurement. The present invention satisfies this need. Tears fulfill anessential role in maintaining ocular surface integrity, protectingagainst microbial challenge, and preserving visual acuity. Thesefunctions in turn, are critically dependent upon the composition andstability of the tear film structure, which includes an underlying mucinfoundation, a middle aqueous component, and an overlying lipid layer.Disruption, deficiency, or absence of the tear film can severely impactthe eye.

SUMMARY

In accordance with the invention, a fluid sample is measured with a tearfilm measuring system that includes a processing device that receives asample chip comprising a sample region configured to contain an aliquotvolume of sample fluid, the processing device configured to performanalyses of osmolarity and of one or more biomarkers within the samplefluid, wherein the analysis of biomarkers includes normalization ofbiomarker concentration values. Processing in accordance with theinvention includes receiving an output signal from a sample region of asample chip that is configured to produce an osmolarity output signalthat indicates energy properties of an aliquot volume of the samplefluid, wherein the osmolarity output signal is correlated withosmolarity of the sample fluid, receiving an output signal from thesample region of the sample chip that is configured to produce abiomarker output signal that indicates chemical properties of the samplefluid, wherein the biomarker output signal is correlated with biomarkerconcentration of the sample fluid, processing the osmolarity outputsignal to produce an osmolarity value for the sample fluid andprocessing the biomarker output signal to produce a biomarkerconcentration value for the sample fluid, and determining an AdjustedBiomarker Level that provides normalization of biomarker concentrationvalues. The normalization of biomarker concentration values can correctfor patient-specific tear homeostasis and for clinician induced tearsampling variance in connection with obtaining the sample fluid. Theprocessing of the osmolarity output signal and processing the biomarkeroutput signal can be performed simultaneously or serially.

These and other features, aspects, and embodiments of the invention aredescribed below in the section entitled “Detailed Description.”

BRIEF DESCRIPTION OF THE DRAWINGS

Features, aspects, and embodiments of the inventions are described inconjunction with the attached drawings, in which:

FIG. 1 illustrates an aliquot-sized sample receiving chip for measuringthe osmolarity of a sample fluid;

FIG. 2 illustrates an alternative embodiment of a sample receiving chipthat includes a circuit region with an array of electrodes imprintedwith photolithography techniques;

FIG. 3 illustrates another alternative embodiment of the FIG. 1 chip,wherein a circuit region includes printed electrodes arranged in aplurality of concentric circles;

FIG. 4 is a top view of the chip shown in FIG. 2;

FIG. 5 is a top view of the chip shown in FIG. 3;

FIG. 6 is a block diagram of an osmolarity measurement system configuredin accordance with the present invention;

FIG. 7 is a perspective view of a tear film osmolarity measurementsystem constructed in accordance with the present invention;

FIG. 8 is a side section of the sample receiving chip showing theopening in the exterior packaging;

FIG. 9 is a calibration curve relating the sodium content of the samplefluid with electrical conductivity;

FIG. 10 illustrates a hinged base unit of the osmometer that utilizesthe sample receiving chips described in FIGS. 1-5;

FIG. 11 illustrates a probe card configuration for the sample receivingchip and processing unit;

FIG. 12 is a flowchart describing an exemplary osmolarity measurementtechnique in accordance with the invention;

FIG. 13 is a flow chart illustrating a method for calibrating anosmolarity measuring system in accordance with another exampleembodiment of the invention;

FIG. 14 is a flow chart illustrating a method for calibrating anosmolarity measuring system in accordance with another exampleembodiment of the invention;

FIG. 15 is a flow chart illustrating a method for calibrating anosmolarity measuring system in accordance with another exampleembodiment of the invention.

FIG. 16 is an image showing residual salt crystals on a miroelectrodearray;

FIG. 17 is a graph illustrating a typical response when a sample fluidis introduced to a microelectrode array;

FIG. 18 is a graph illustrating a response when a sample fluid isintroduced to a microelectrode array that contains residual salt; and

FIG. 19 is a flow chart illustrating a method for calibrating anosmolarity measuring system in accordance with another exampleembodiment of the invention.

FIG. 20 is a flow chart illustrating biomarker normalization inaccordance with the invention.

FIG. 21 is a plan view of a receiving substrate in which osmolarity ismultiplexed in space with biomarker detection.

FIG. 22 is a detail view of the FIG. 21 receiving substrate showing thearrangement of electrodes in the sample region.

DETAILED DESCRIPTION

Exemplary embodiments are described for measuring the osmolarity of analiquot volume of a sample fluid (e.g., tear film, sweat, blood, orother fluids). The exemplary embodiments are configured to be relativelyfast, non-invasive, inexpensive, and easy to use, with minimal injury ofrisk to the patient. Accurate measurements can be provided with aslittle as nanoliter volumes of a sample fluid. For example, a measuringdevice configured in accordance with the invention enables osmolaritymeasurement with no more than 200 μL of sample fluid, and typically muchsmaller volumes can be successfully measured. In one embodimentdescribed further below, osmolarity measurement accuracy is notcompromised by variations in the volume of sample fluid collected, sothat osmolarity measurement is substantially independent of collectedvolume. The sample fluid can include tear film, sweat, blood, or otherbodily fluids. It should be noted, however, that sample fluid cancomprise other fluids, such as milk or other beverages.

FIG. 1 illustrates an exemplary embodiment of an osmolarity chip 100that can be used to measure the osmolarity of a sample fluid 102, suchas a tear film sample. In the FIG. 1 embodiment, the chip 100 includes asubstrate 104 with a sample region having sensor electrodes 108, 109 andcircuit connections 110 imprinted on the substrate. The electrodes andcircuit connections are preferably printed using well-knownphotolithographic techniques. For example, current techniques enable theelectrodes 108, 109 to have a diameter in the range of approximately one(1) to eighty (80) microns, and spaced apart sufficiently so that noconductive path exists in the absence of sample fluid. Currentlyavailable techniques, however, can provide electrodes of less than onemicron in diameter, and these are sufficient for a chip constructed inaccordance with the invention. The amount of sample fluid needed formeasurement is no more than is necessary to extend from one electrode tothe other, thereby providing an operative conductive path. Thephotolithographic scale of the chip 100 permits the measurement to bemade for aliquot-sized samples in a micro- or nano-scale level. Forexample, reliable osmolarity measurement can be obtained with a samplevolume of less than 20 μL of tear film. A typical sample volume is lessthan one hundred nanoliters (100 nL). It is expected that it will berelatively easy to collect 10 nL of a tear film sample even frompatients suffering from dry eye.

The chip 100 is configured to transfer energy to the sample fluid 102and enable detection of the sample fluid energy properties. In thisregard, a current source is applied across the electrodes 108, 109through the connections 110. The osmolarity of the sample fluid can bemeasured by sensing the energy transfer properties of the sample fluid102. The energy transfer properties can include, for example, electricalconductivity, such that the impedance of the sample fluid is measured,given a particular amount of electrical power (e.g., current) that istransferred into the sample through the connections 110 and theelectrodes 108, 109.

If conductivity of the sample fluid is to be measured, then preferably asinusoidal signal on the order of ten volts at approximately 100 kHz isapplied. The real and imaginary parts of the complex impedance of thecircuit path from one electrode 108 through the sample fluid 102 to theother electrode 109 are measured. At the frequencies of interest, it islikely that the majority of the electrical signal will be in the realhalf of the complex plane, which reduces to the conductivity of thesample fluid. This electrical signal (hereafter referred to asconductivity) can be directly related to the ion concentration of thesample fluid 102, and the osmolarity can be determined. Moreover, if theion concentration of the sample fluid 102 changes, the electricalconductivity and the osmolarity of the fluid will change in acorresponding manner. Therefore, the osmolarity is reliably obtained. Inaddition, because the impedance value does not depend on the volume ofthe sample fluid 102, the osmolarity measurement can be madesubstantially independent of the sample volume.

As an alternative to the input signal described above, more complexsignals can be applied to the sample fluid whose response willcontribute to a more thorough estimate of osmolarity. For example,calibration can be achieved by measuring impedances over a range offrequencies. These impedances can be either simultaneously (via combinedwaveform input and Fourier decomposition) or sequentially measured. Thefrequency versus impedance data will provide information about thesample and the relative performance of the sample fluid measurementcircuit.

FIG. 2 illustrates an alternative embodiment of a sample receiving chip200 that measures osmolarity of a sample fluid 202, wherein the chipcomprises a substrate layer 204 with a sample region 206 comprising animprinted circuit that includes an array of electrodes 208. In theillustrated embodiment of FIG. 2, the sample region 206 has a 5-by-5array of electrodes that are imprinted with photolithographictechniques, with each electrode 208 having a connection 210 to one sideof the substrate 204. Not all of the electrodes 208 in FIG. 2 are shownwith a connection, for simplicity of illustration. The electrodesprovide measurements to a separate processing unit, described furtherbelow.

The electrode array of FIG. 2 provides a means to measure the size ofthe tear droplet 202 by detecting the extent of conducting electrodes208 to thereby determine the extent of the droplet. In particular,processing circuitry can determine the number of electrodes that areconducting, and therefore the number of adjacent electrodes that arecovered by the droplet 202 will be determined. The planar area of thesubstrate that is covered by the sample fluid is thereby determined.With a known nominal surface tension of the sample fluid, the height ofthe sample fluid volume over the planar area can be reliably estimated,and therefore the volume of the droplet 202 can be determined.

FIG. 3 illustrates another alternative embodiment of a sample receivingchip 300 on which a sample fluid 302 is deposited. The chip comprises asubstrate layer 304, wherein a sample region 306 is provided withelectrodes 308 that are configured in a plurality of concentric circles.Each electrode 308 can be connected to one side of substrate layer 304by connections 310. In a manner similar to the square array of FIG. 2,the circular arrangement of the FIG. 3 electrodes 308 also provides anestimate of the size of the sample fluid volume 302 because the droplettypically covers a circular or oval area of the sample region 302.Processing circuitry can detect the largest (outermost) circle ofelectrodes that are conducting, and thereby determine a planar area ofcoverage by the fluid sample. As before, the determined planar areaprovides a volume estimate, in conjunction with a known surface tensionand corresponding volume height of the sample fluid 302. In the FIG. 3illustrated embodiment, the electrodes 308 can be printed using wellknown photolithography techniques that currently permit electrodes tohave a diameter in the range of one (1) to eighty (80) microns. Thisallows the submicroliter droplet to substantially cover the electrodes.The electrodes can be printed over an area sized to receive the samplefluid, generally covering 1 mm² to 1 cm².

The electrodes and connections shown in FIG. 1, FIG. 2, and FIG. 3 canbe imprinted on the respective substrate layers as electrodes withcontact pads, using photolithographic techniques. For example, theelectrodes can be formed with different conductive metalization such asaluminum, platinum, titanium, titanium-tungsten, and other similarmaterial. In one embodiment, the electrodes can be formed with adielectric rim to protect field densities at the edges of theelectrodes. This can reduce an otherwise unstable electric field at therim of the electrode.

Top views of the exemplary embodiments of the chips 200 and 300 areillustrated in FIG. 4 and FIG. 5, respectively. The embodiments show thedetailed layout of the electrodes and the connections, and illustratehow each electrode can be electrically connected for measuring theelectrical properties of a sample droplet. As mentioned above, thelayout of the electrodes and the connections can be imprinted on thesubstrate 100, 200, 300 using well-known photolithographic techniques.

FIG. 6 is a block diagram of an osmometry system 600 configured inaccordance with an embodiment of the present invention, showing howinformation is determined and used in a process that determinesosmolarity of a sample fluid. The osmometry system 600 includes ameasurement device 604 and a processing device 606. The measurementdevice receives a volume of sample fluid from a collection device 608.The collection device can comprise, for example, a micropipette orcapillary tube. The collection device 608 collects a sample tear film ofa patient, such as by using negative pressure from a fixed-volumemicropipette or charge attraction from a capillary tube to draw a smalltear volume from the vicinity of the ocular surface of a patient.

The measurement device 604 can comprise a system that transfers energyto the fluid in the sample region and detects the imparted energy. Forexample, the measurement device 604 can comprise circuitry that provideselectrical energy in a specified waveform (such as from a functiongenerator) to the electrical path comprising two electrodes bridged bythe sample fluid. The processing device 606 detects the energy impartedto the sample fluid and determines osmolarity. The processing device cancomprise, for example, a system including an RLC multimeter thatproduces data relating to the reactance of the fluid that forms theconductive path between two electrodes, and including a processor thatdetermines osmolarity through a table look-up scheme. If desired, theprocessing device can be housed in a base unit that receives one of thechips described above.

As mentioned above, a sample sufficient to provide an osmolaritymeasurement can contain less than 20 microliters (μL) of fluid. Atypical sample of tear film in accordance with the invention iscollected by a fluid collector such as a capillary tube, which oftencontains less than one microliter of tear film. Medical professionalswill be familiar with the use of micropipettes and capillary tubes, andwill be able to easily collect the small sample volumes describedherein, even in the case of dry eye sufferers.

The collected sample fluid is expelled from the collection device 608 tothe measurement device 604. The collection device can be positionedabove the sample region of the chip substrate either manually by amedical professional or by being mechanically guided over the sampleregion. In one embodiment, for example, the collection device (e.g., acapillary tube) is mechanically guided into position with aninjection-molded plastic hole in a base unit, or is fitted to a set ofclamps with precision screws (e.g., a micromanipulator with needles formicrochip interfaces). In another embodiment, the guide is acomputer-guided feedback control circuitry that holds the capillary tubeand automatically lowers it into the proper position.

The electrodes and connections of the chips measure energy properties ofthe sample fluid, such as conductivity, and enable the measuredproperties to received by the processing device 606. The measured energyproperties of the sample fluid include electrical conductivity and canalso include other parameters, such as both parts of the compleximpedance of the sample, the variance of the noise in the output signal,and the measurement drift due to resistive heating of the sample fluid.The measured energy properties are processed in the processing device606 to provide the osmolarity of the sample. In one embodiment, theprocessing device 606 comprises a base unit that can accept a chip andcan provide electrical connection between the chip and the processingdevice 606. In another embodiment, the base unit can include a displayunit for displaying osmolarity values. It should be noted that theprocessing device 606 and, in particular, the base unit can be ahand-held unit.

FIG. 7 is a perspective view of a tear film osmolarity measuring system700 constructed in accordance with the present invention. In theillustrated embodiment of FIG. 7, the exemplary system 700 includes ameasuring unit 701 that comprises a chip, such as one of the chipsdescribed above, and a connector or socket base 710, which provides theappropriate measurement output. The system 700 determines osmolarity bymeasuring electrical conductivity of the sample fluid: Therefore, themeasurement chip 701 comprises a semiconductor integrated circuit (IC)chip with a substrate having a construction similar to that of the chipsdescribed above in connection with FIG. 1 through FIG. 5. Thus, the chip701 includes a substrate layer with a sample region that is defined byat least two electrodes printed onto the substrate layer (such detailsare of a scale too small to be visible in FIG. 7; see FIG. 1 throughFIG. 5). The substrate and sample region are encased within an inertpackage, in a manner that will be known to those skilled in the art. Inparticular, the chip 701 is fabricated using conventional semiconductorfabrication techniques into an IC package 707 that includes electricalconnection legs 708 that permit electrical signals to be received by thechip 701 and output to be communicated outside of the chip. Thepackaging 707 provides a casing that makes handling of the chip moreconvenient and helps reduce evaporation of the sample fluid.

FIG. 8 shows that the measurement chip 701 is fabricated with anexterior opening hole 720 into which the sample fluid 702 is inserted.Thus, the hole 720 can be formed in the semiconductor packaging 707 toprovide a path through the chip exterior to the substrate 804 and thesample region 806. The collection device (such as a micropipette orcapillary tube) 808 is positioned into the hole 720 such that the samplefluid 702 is expelled from the collection device directly onto thesample region 806 of the substrate 804. The hole 720 is sized to receivethe tip of the collection device. The hole 720 forms an opening orfunnel that leads from the exterior of the chip onto the sample region806 of the substrate 804. In this way, the sample fluid 702 is expelledfrom the collection device 808 and is deposited directly on the sampleregion 806 of the substrate 804. The sample region is sized to receivethe volume of sample fluid from the collection device. In FIG. 8, forexample, the electrodes form a sample region 806 that is generally in arange of approximately 1 mm² to 1 cm² in area.

Returning to FIG. 7, the chip 701 can include processing circuitry 704that comprises, for example, a function generator that generates asignal of a desired waveform, which is applied to the sample regionelectrodes of the chip, and a voltage measuring device to measure theroot-mean-square (RMS) voltage value that is read from the chipelectrodes. The function generator can produce high frequencyalternating current (AC) to avoid undesirable direct current (DC)effects for the measurement process. The voltage measuring device canincorporate the functionality of an RLC measuring device. Thus, the chip701 can incorporate the measurement circuitry as well as the sampleregion electrodes. The processing circuitry can include a centralprocessing unit (CPU) and associated memory that can store programminginstructions (such as firmware) and also can store data. In this way, asingle chip can include the electrodes and associated connections forthe sample region, and on a separate region of the chip, can alsoinclude the measurement circuitry. This configuration will minimize theassociated stray resistances of the circuit structures.

As noted above, the processing circuitry 70 applies a signal waveform tothe sample region electrodes. The processing circuitry also receives theenergy property signals from the electrodes and determines theosmolarity value of the sample fluid. For example, the processing unitreceives electrical conductivity values from a set of electrode pairs.Those skilled in the art will be familiar with techniques and circuitryfor determining the conductivity of a sample fluid that forms aconducting path between two or more electrodes.

In the FIG. 7 embodiment, the processing unit 704 produces signalwaveforms at a single frequency, such as 100 kHz and 10 Voltspeak-to-peak. The processing circuitry 704 then determines theosmolarity value from the sodium content correlated to the electricalconductivity using a calibration curve, such as the curve shown in FIG.9. In this case, the calibration curve is constructed as a transferfunction between the electrical conductivity (voltage) and theosmolarity value (i.e., the sodium content). It should be noted,however, that other calibration curves can also be constructed toprovide transfer functions between other energy properties and theosmolarity value. For example, the variance, autocorrelation and driftof the signal can be included in an osmolarity calculation. If desired,the osmolarity value can also be built upon multi-variable correlationcoefficient charts or neural network interpretation so that theosmolarity value can be optimized with an arbitrarily large set ofmeasured variables.

In an alternate form of the FIG. 7 embodiment, the processing unit 704produces signal waveforms of a predetermined frequency sweep, such as 1kHz to 100 kHz in 1 kHz increments, and stores the conductivity andvariance values received from the set of electrode pairs at eachfrequency. The output signal versus frequency curve can then be used toprovide higher order information about the sample which can be used withthe aforementioned transfer functions to produce an ideal osmolarityreading.

As shown in FIG. 7, the base socket connector 710 receives the pins 708of the chip 701 into corresponding sockets 711. The connector 710, forexample, can supply the requisite electrical power to the processingcircuitry 704 and electrodes of the chip. Thus, the chip 701 can includethe sample region electrodes and the signal generator and processingcircuitry necessary for determining osmolarity, and the outputcomprising the osmolarity value can be communicated off the chip via thepins 708 through the connector 710 and to a display readout.

If desired, the base connector socket 710 can include a Peltier layer712 located beneath the sockets that receive the pins 708 of the chip701. Those skilled in the art will understand that a Peltier layercomprises an electrical/ceramic junction such that properly appliedcurrent can cool or heat the Peltier layer. In this way, the sample chip701 can be heated or cooled, thereby further controlling evaporation ofthe sample fluid. It should be apparent that evaporation of the samplefluid should be carefully controlled, to ensure accurate osmolarityvalues obtained from the sample fluid.

FIG. 10 shows an alternative embodiment of an osmometer in which thechip does not include an on-chip processing unit such as describedabove, but rather includes limited circuitry comprising primarily thesample region electrodes and interconnections. That is, the processingunit is separately located from the chip and can be provided in the baseunit.

FIG. 10 shows in detail an osmometer 1000 that includes a base unit1004, which houses the base connector 710, and a hinged cover 1006 thatcloses over the base connector 710 and a received measurement chip 701.Thus, after the sample fluid has been dispensed on the chip, the chip isinserted into the socket connector 710 of the base unit 1004 and thehinged cover 1006 is closed over the chip to reduce the rate ofevaporation of the sample fluid.

It should be noted that the problem with relatively fast evaporation ofthe sample fluid can generally be handled in one of two ways. One way isto measure the sample fluid voltage quickly as soon possible after thedroplet is placed on the sample region of the chip. Another way is toenable the measuring unit to measure the rate of evaporation along withthe corresponding changes in conductivity values. The processing unitcan then post-process the output to estimate the osmolarity value. Theprocessing can be performed in the hardware or in software stored in thehardware. Thus, the processing unit can incorporate different processingtechniques such as using neural networks to collect and learn aboutcharacteristic of the fluid samples being measured for osmolarity, aswell as temperature variations, volume changes, and other relatedparameters so that the system can be trained in accordance with neuralnetwork techniques to make faster and more accurate osmolaritymeasurements.

FIG. 11 shows another alternative construction, in which the osmolaritysystem utilizes a sample receiving chip 1102 that does not include ICpackaging such as shown in FIG. 7. Rather, the FIG. 11 measurement chip1102 is configured as a chip with an exposed sample region comprisingthe electrodes and associated connections, but the processing circuitryis located in the base unit for measuring the energy properties of thesample fluid. In this alternative construction, a connector similar tothe connector socket 710 allows transmission of measured energyproperties to the processing unit in the base unit. Those skilled in theart will understand that such a configuration is commonly referred to aprobe card structure.

FIG. 11 shows a probe card base unit 1100 that receives a sample chipprobe card 1102 that comprises a substrate 1104 with a sample region1106 on which are formed electrodes 1108 that are wire bonded to edgeconnectors 1110 of the probe card. When the hinged lid 1112 of the baseunit is closed down over the probe card, connecting tines 1114 on theunderside of the lid come into mating contact with the edge connectors1110. In this way, the electrodes of the sample region 1106 are coupledto the processing circuitry and measurement can take place. Theprocessing circuitry of the probe card embodiment of FIG. 11 can beconfigured in either of the configurations described above. That is, theprocessing to apply current to the electrodes and to detect energyproperties of the sample fluid and determine osmolarity can be locatedon-chip, on the substrate of the probe card 1102, or the processingcircuitry can be located off-chip, in the base unit 1100.

In all the alternative embodiments described above, the osmometer isused by placing a new measurement chip into the base unit while thehinged top is open. Upon placement into the base unit the chip islowered up and begins monitoring its environment. Recording outputsignals from the chip at a rate of, for example, 1 kHz, will fullycapture the behavior of the system. Placing a sample onto any portion ofthe electrode array generates high signal-to-noise increase inconductivity between ally pair of electrodes covered by the samplefluid. The processing unit will recognize the change in conductivity asbeing directly related to the addition of sample fluid, and will beginconversion of electronic signals into osmolarity data once this type ofchange is identified. This strategy occurs without intervention bymedical professionals. That is, the chip processing is initiated uponcoupling to the base unit and is not dependent on operating the lid ofthe base unit or any other user intervention.

In any of the configurations described above, either the “smart chip”with processing circuitry on-chip (FIG. 7), or the electrode-onlyconfiguration with processing circuitry off-chip (FIG. 10), in apackaged chip (FIG. 7 and FIG. 10) or in a probe card (FIG. 1), thesample receiving chip can be disposed of after each use, so that thebase unit serves as a platform for interfacing with the disposablemeasurement chip. As noted, the base unit can also include relevantcontrol, communication, and display circuits (not shown), as well assoftware, or such features can be provided off-chip in the base unit. Inthis regard, the processing circuitry can be configured to automaticallyprovide sufficient power to the sample region electrodes to irreversiblyoxidize them after a measurement cycle, such that the electrodes arerendered inoperable for any subsequent measurement cycle. Upon inserteda used chip into the base unit, the user will be given an indicationthat the electrodes are inoperable. This helps prevent inadvertentmultiple use of a sample chip, which can lead to inaccurate osmolarityreadings and potentially unsanitary conditions.

A secondary approach to ensure that a previously used chip is not placedback into the machine includes encoding serial numbers, or codesdirectly onto the chip. The base unit will store the used chip numbersin memory and cross-reference them against new chips placed in the baseconnector. If the base unit finds that the serial number of the usedchip is the same as an old chip, then the system will refuse to measureosmolarity until a new chip is inserted. It is important to ensure useof a new chip for each test because proteins adsorb and salt crystalsform on the electrodes after evaporation has run its course, whichcorrupt the integrity of the measuring electrodes.

FIG. 12 is a flowchart describing an exemplary (osmolarity measurementtechnique in accordance with the invention. A body fluid sample, such asa tear film, is collected at box 1300. The sample typically containsless than one microliter. At box 1302, the collected sample is depositedon a sample region of the chip substrate. The energy properties of thesample are then measured at box 1304. The measured energy properties arethen processed, at box 1306, to determine the osmolarity of the sample.If the chip operates in accordance with electrical conductivitymeasurement, then the measurement processing at box 1306 can include the“electrode oxidation” operation described above that renders the chipelectrodes inoperable for any subsequent measuring cycles.

In the measurement process for a conductivity measuring system, asubstantially instantaneous shift is observed from the open circuitvoltage to a value that closely represents the state of the sample atthe time of collection, upon placement of a sample tear film on anelectrode array of the substrate. Subsequently, a drift in theconductivity of the sample will be reflected as a continual change inthe output.

The output of the measurement chip can be a time-varying voltage that istranslated into an osmolarity value. Thus, in a conductivity-basedsystem, more information than just the “electrical conductivity” of thesample can be obtained by measuring the frequency response over a widerange of input signals, which improves the end stage processing. Forexample, the calibration can be made over a multiple frequencies (e.g.,measure ratio of signals at 10, 20, 30, 40, 50, 100 Hz) to make themeasurement process a relative calculation. This makes the chip-to-chipvoltage drift small. The standard method for macroscale electrode basedmeasurements (i.e. in a pH meter, or microcapillary technique) is torely upon known buffers to set up a linear calibration curve. Becausephotolithography is a relatively reproducible manufacturing technique,when coupled to a frequency sweep, calibration can be performed withoutoperator intervention.

As mentioned above, the processing of the energy properties can beperformed in a neural network configuration, where the seeminglydisparate measured data points obtained from the energy properties canbe used to provide more accurate osmolarity reading than from a singleenergy property measurement. For example, if only the electricalconductivity of the sample is measured, then the calibration curve canbe used to simply obtain the osmolarity value corresponding to theconductivity. This osmolarity value, however, generally will not be asaccurate as the output of the neural network.

The neural network can be designed to operate on a collection ofcalibration curves that reflects a substantially optimized transferfunction between the energy properties of the sample fluid and theosmolarity. Thus, in one embodiment, the neural network constructs acollection of calibration curves for all variables of interest, such asvoltage, evaporation rate and volume change. The neural network can alsoconstruct or receive as an input a priority list that assigns animportance factor to each variable to indicate the importance of thevariable to the final outcome, or the osmolarity value. The neuralnetwork constructs the calibration curves by training on examples ofreal data where the final outcome is known a priori. Accordingly, theneural network will be trained to predict the final outcome from thebest possible combination of variables. This neural networkconfiguration that processes the variables in an efficient combinationis then loaded into the processing unit residing in the measurement chip701 or the base unit. Once trained, the neural network can be configuredin software or hardware.

The ability to identify and subtract out manufacturing defects in theelectrodes prior to osmolarity testing can also be important. This toocan be accomplished via calibration of an osmolarity calibration devicethat comprises an osmolarity chip, such as chip 1200 illustrated in FIG.2. This type of calibration can also be achieved, possibly in a moreefficient manner, through the use of neural networks, but it will beunderstood that such networks are not necessary to achieve thecalibration processes described in the following description.

Classically, bare metal electrodes were considered poor measuringdevices when placed in direct contact with an electrochemical solutionof interest. Foremost, there can exist a double layer of counter ionsthat surround the electrode at the metal/solution interface that canimpose a field of sufficient magnitude to significantly alter the ionquantity of interest. In bulk solutions, convection currents or stirringcan disrupt these distributions and cause time varying noise, whosemagnitude is on the order of the relevant signal. Further, thepolarizability and hysterisis of the electrodes can cause problems ifsourcing small signals to the electrodes. Finally, large DC or lowfrequency AC sources from the electrode can also cause irreversibleelectrolysis that results in bubbling and oxidation of importantbiological species. Bubbles introduce variable dielectric shifts nearthe electrode and invalidate inferences drawn about the solution fromvoltages measured under such conditions.

A conventional solution for these effects is to physically separate theelectrodes from the solution through a salt bridge, whereupon bubblingand other nonlinearities in the immediate vicinity of the activeelectrodes are largely irrelevant to the steady state distribution ofions that flow far from the electrodes. As an example, in devicesconfigured to measure the pH of a solution, the metal electrodes can beseparated from the bulk solution with a semipermeable membrane such asglass or ceramic material. Moreover, the metal electrode within thesemipermeable membrane is generally comprised of Ag (silver) or calomel(mercury) immersed in a silver chloride or mercurous chloride solution.This allows a single chemical reaction to dominate action close to theelectrode. The reaction can be kept close to equilibrium, and when agradient of ions is created across the semipermeable membrane, anosmotic force is transmitted to the electrode surface through thesymmetric redox reaction which drives the system back towardsequilibrium. In this way, ions are balanced at the glass-solution andelectrode-buffer interface, and nonlinearities can be minimized.

In contrast to typical measurement systems, however, clinicalmeasurements of human tear film osmolarity require far smallerelectrodes than traditional systems. This is due to the fact that tensof nanoliters represent the maximum viable collection volume frompatients with, e.g., keratoconjunctivitis sicca. As described above, thesystems and methods described herein can allow for a clinical device fortear measurements that can meet the strict requirements for accuratemeasurements and diagnosis in this area by using bare metal electrodesprinted on a microchip, e.g., as shown in FIG. 1 and FIG. 2. As aresult, none of the traditional solutions to electrode shielding arefeasible for such devices because the physical dimensions are far toosmall. At, for example, 80 μm in diameter, the photolithographedelectrodes preclude membranes from being manufactured in a costeffective manner. For example, a spin coated gel permeation layer isprohibitively expensive, results in a low yield process, and introducesseveral manufacturing variances. Further, osmotic perturbations due tosalt bridge gradients would overwhelm the minuscule sample volume ofinterest. Therefore, many of the typical methods for taking measurementswith macroscale electrodes cannot be applied to microelectrodes, andadditional issues of calibration remain.

In order to establish a linear calibration profile, where input directlyscales with output, conventional macroscale electrodes are typicallyimmersed in multiple known standards. For instance, pH meters will use aset of three buffers at pH 4, 7 and 10, with each fluid marking a pointfor the fitted line. Between each calibration point, the macroscaleelectrode can be washed and dried to ensure that the standards do notmix. If one assumes that the electrode buffer inside the glass chamberis of a certain concentration, then calibration can be performed with aslittle as one standard point. Over time however, the once homogeneouselectrode buffer becomes contaminated with the substances it hasmeasured, which then requires at least a two-point calibration in orderto be precise.

When working with microelectrodes, however, conventional calibrationsteps, such as those described above, are often impossible to performwithout risking damage to the array and compromising any ensuingmeasurements. For instance after a calibration standard has been placedon the chip, it is impractical to clean the array with paper, becausescratches on the electrode surface will result in exceedingly highcurrent densities at the scratch edge, which then leads to bubbling andinvalid measurements. Furthermore, if one were to use a model of humantears for the calibration standard, i.e. with 10 mg/ml BSA as aconstituent, protein adsorption to the electrode surface would corruptthe purity of a clinical measurement. Finally, if a small amount of saltsolution was used to set calibration points, the fluid would evaporate,leaving a very noticeable salt crystal upon random parts of the chipsurface. This residual salt will then dissolve into any subsequentsample that is placed on the chip, and unlike the volume independencedisplayed by conductivity, slight differences in the amount of fluiddeposited as a standard will result in different amounts of salt addedto the fluid sample of interest.

Ultimately, a clinical test for dry eye requires a conversion from therelative motion of ions in solution to an absolute osmolarity that canbe compared between tests over time. The final value must be independentof the measuring device and stable over time to qualify for diagnosticpurposes. Accordingly, the ability to calibrate a microelectrode array,such as those described above, can be hampered by several remainingchallenges when attempting to obtain the strictest possible tolerancesfor the measuring device.

As described below, however, several approaches can be used to calibratea microelectrode array, such as those described above, in accordancewith the systems and methods described herein. These approaches can eachstart by determining an intrinsic conductivity for each electrode in thearray. This intrinsic conductivity can then be stored and used tosubtract out the effect of the intrinsic conductivity form finalmeasurements of the electrical properties of a test fluid, such as atear. Depending on the embodiment, a standard may or may not be used indetermining a calibration factor for the electrodes. Further, when astandard is used, a subsequent washing step may or may not be included.

It should also be pointed out that the various approaches can becombined in a modular fashion to produce ever more accurate calibrationresults. The approaches can thus be used tiered to produce successivelevels of intricacy in order to minimize variability between tests.

FIG. 13 is a flow chart that illustrates one embodiment of a method forcalibrating an osmolarity measuring device that does not use a standardin accordance with one embodiment of the systems and methods describedherein. At box 1402, the intrinsic conductivity of the electrodes ismeasured. The measured intrinsic conductivity for each electrode canthen be stored on a memory. At box 1404, a sample fluid of interest,such as a tear film, is introduced to the measuring device, and theelectrical properties of the sample fluid are measured at box 1406. Inone embodiment, the processing circuitry also identifies the electrodesfrom the electrode array that are in contact with the sample fluid atbox 1408. The electrodes that are in contact with the sample fluid areconducting electrodes, and the identity of the conducting electrodes canalso be stored in memory. At box 1410, the processing circuitry adjuststhe measured electrical properties of the sample fluid to adjust for theintrinsic conductivity of the electrodes, on a pair-wise basis, thatperformed the measurement of the sample. This adjustment results anosmolarity measurement of the sample alone, and is independent ofvariances in the thickness of electrode metalization, dielectricdeposition, and other variances in the electrodes that can occur duringmanufacturing of the measuring device.

The intrinsic conductivity can be determined (box 1402), in oneembodiment, by applying a DC current to the electrode array andmeasuring the resulting output voltage for each electrode. Thecorresponding resistance can then be calculated based on the DC currentand the output voltage and, e.g., stored in memory. In an alternativeembodiment, a more complex signal, e.g., a sine wave, can be generatedin the time domain and applied to the array of electrodes. Thecorresponding outputs can then be measured and stored. A Fouriertransform can then be applied to the stored output data. The result is amap of amplitude versus frequency that indicates the relativeconductance over a range of frequencies for each electrode. This map canbe generated for a range of frequencies of interest for a particularimplementation, e.g., from the low kHz to the MHz range.

In order to deliver a current signal to each electrode and measure theresulting output for calibration purposes, two leads can be provided foreach electrode. The current signal can then be applied and the outputmeasured, for a given electrode, via the two leads.

In such an embodiment, the slope of the resulting calibration curve canbe assumed to be constant over time. The curve can then be built into aosmolarity measurement device, such as those described above.Adjustments to the osmolarity determinations can then be made throughelectrode resistance subtraction, which will simply shift the inputmapping along the x-axis of the calibration curve. Other effects, suchas humidity and ambient temperature effects can then, depending on theembodiment, be accounted for in subsequent signal processing.

The ability to map out the intrinsic conductivity of each electrode pairprior to testing also gives a confidence bound to the array locations.In this manner, the electrode is defined as a random process of gaussianrandom variables with a sample mean and variance as defined by theconductivity calculations above. Any electrode outside the 95thpercentile of the expected variance can be considered flawed, and itssignals can be neglected in future calculations. This ability toselectively address electrode pairs in an array enhances the ability tocalibrate the reading and protect against spurious manufacturingdefects.

As an example, it should be remembered that the electrode array of FIG.2 provides a means to measure the size of, e.g., a tear droplet 202 bydetecting the extent of conducting electrodes 208 to thereby determinethe extent of the droplet. In particular, processing circuitry candetermine the number of electrodes that are conducting, and thereforethe number of adjacent electrodes that are covered by the droplet 202.The identities of the electrodes from the array that are conducting andin contact with the sample fluid 202 can then stored in memory.

Thus, following the completion of the sample testing, the intrinsicconductivity of all of the conducting electrode pairs can be subtractedfrom the sample output signal to calculate an osmolarity valueindicative of the sample alone. In one embodiment, it is important torecognize that the sample fluid 202 will not cover all electrodes in thearray. Therefore, only those electrodes that are conducting and incontact with the sample fluid 202 are included in the calculation toadjust the sample measurement. As mentioned, the resulting osmolaritymeasurement of the sample fluid 202 is therefore made independent ofvariances in the thickness of electrode metalization, dielectricdeposition, and other variances that may occur during manufacturing forthe conducting electrodes that perform the measurement.

While the systems and methods from calibration just described are usefuland simple to implement, requiring minimal software post processing toaccomplish any needed correction, it is unlikely that this method willdetect sharp deformities in electrode geometries such as metal peaks orrough edges because these defects will not significantly alter theintrinsic conductivity of the electrodes. It can be shown that the baremetal electrodes described above suffice for measurement when highfrequency sine waves are used as input signals to the microelectrodes.Even at 10 V peak to peak, 10-100 kHz waves do not initiate bubbling inthe aliquot of tear film sample that is being measured. This is can bedue to the fact that within this frequency range, there is a balancebetween water polarizability and ionic mobility, resulting inoscillations of ions rather than bulk movement. This solves manyproblems with electrolysis and other DC electrode problems. However,when a sample fluid is applied, electrode geometries such as metal peaksor rough edges may cause bubbling and mar the measurement. Therefore, inorder to account for these effects, it is useful to begin each test witha one- or two-points standard calibration prior to use.

FIG. 14 is a flow chart that illustrates one embodiment of a method forcalibrating an osmolarity measuring device with a standard fluid. At box1502, a calibration curve is provided on a memory, and the calibrationcurve is assumed to be a straight line. One point of the line isobtained through the assumption that when the measured electricalproperties of the standard are equal to zero, the osmolarity of thestandard is equal to zero. The electrical properties (i.e. sine waveFourier transform, etc.) of the high end of the concentration range,around 500 mOsms, can then be predefined in memory based on knownelectrical properties for a fluid having a known concentration.

At box 1504, a standard fluid can be deposited onto the microelectrodearray of a measuring device, and the electrical properties of thestandard can be measured at box 1504. The methods for measuring theelectrical properties of the standard fluid can include the methodsdescribed above for measuring the electrical properties of a samplefluid. A processing device can then be configured to correlate themeasured electrical properties to an osmolarity value and, e.g., storethe osmolarity measurement of the standard fluid in memory.

In one embodiment, the standard fluid that is added at box 1504comprises a small aliquot, for example, 1 μL, of deionized water. Theosmolarity measurement for deionized water can be registered as a lowerbound on the calibration curve since deionized water exhibits a minimumamount of osmotic character. In one embodiment, a one-point calibrationis used such that the entire range for the measurement scale of thedevice can be extrapolated based on the difference between the expectedosmolarity of deionized water and the actual measured osmolarity of thestandard. At box 1506, the processing device determines a calibrationfactor to adjust the slope of the measurement scale to match thecalibration curve. Further, any adjustments to the slope of themeasurement scale are made with the measured fluid per electrode pair,such that the final value from each electrode pair is equivalent withall others. The final calibration factor can then be stored in memory.

After calibration has been determined, the standard can be allowed toevaporate from the microelectrode array at box 1508. Evaporation can benecessary to prevent the standard from mixing with and corrupting thesample fluid. Deionized water provides an exemplary standard when thedeionized water has such low salt content that there is no salt crystaldeposited on the chip after evaporation.

In one embodiment where there is no salt crystal remaining after thestandard evaporates, the sample fluid to be tested can then be depositedon the microelectrode array of the measuring device at box 1510. Themicroelectrode array transfers energy to the sample fluid and enablesthe detection of the sample fluid's electrical properties, which aremapped to an osmolarity measurement at box 1512 as described above. Atbox 1514, a processing device can be configured to adjust the osmolaritymeasurement based on the previously determined calibration factor. Theuse of the calibration factor results in an osmolarity measurement thatis substantially independent from variances in the geometry of themicroelectrode array.

The process of FIG. 14 can also be combined with the simpler process ofFIG. 13 in order to improve accuracy.

FIG. 15 is a flow chart that illustrates another embodiment of a methodfor calibrating an osmolarity measuring device using a standard fluidthat is a slat solution. At box 1602, a calibration curve can beprovided on a memory, and the calibration curve can be assumed to be astraight line. One point of the line is obtained through the assumptionthat when the measured electrical properties of the standard are equalto zero, the osmolarity of the standard is equal to zero. The electricalproperties of the high end of the concentration range, around 500 mOsms,can be predefined in memory based on known electrical properties for afluid having a known concentration.

At box 1604 a standard fluid can then be deposited onto themicroelectrode array of a measuring device, and the electricalproperties of the standard can be measured. The methods for measuringthe electrical properties of the standard can, for example, include themethods described above for measuring the electrical properties of asample fluid. A processing device can then be configured to correlatethe measured electrical properties to an osmolarity value, and store theosmolarity measurement of the standard on a memory.

At box 1606, the processing device can be configured to then determine acalibration factor to adjust the slope of the measurement scale to matchthe calibration curve. Further, any adjustments to the slope of themeasurement scale can be made with the measured fluid on a per electrodepair basis, such that the final value from each electrode pair isequivalent with all others. The final calibration factor can then bestored in memory.

In this process, however, the standard can be a simple salt solution(NaCl), or a complex salt solution, e.g., with sodium, potassium,calcium and magnesium salts in physiological ratios. However, when thesalt solution evaporates at box 1608, a very noticeable salt crystalwill often remain on the chip surface as shown in FIG. 16. When thisoccurs, the left over salt crystal should be accounted for in thesubsequent osmolarity measurement that is made at box 1612.

For example, FIG. 17 demonstrates a typical response when a sample fluidis introduced to a microelectrode array that does not include residualsalt. In comparison, FIG. 18 shows the response when residual salt ispresent on the microelectrode array at the time the sample fluid isintroduced. The presence of a salt crystal clearly alters the response,such that it steadily declines for a period before righting itself andheading into the steady evaporation mode. As shown in FIG. 18, thenormal second order dynamics are suppressed. This is due to the factthat upon sample placement, the residual salt crystal will begin todissolve into the sample fluid. The concentration of residual salt nearthe electrode will continue to decrease until its contribution hasbecome uniformly mixed throughout the sample, whereupon the curve willbegin to rise again due to evaporation.

During this transient response, dissolving ions between two measuringelectrodes will present a much higher conductivity than in theoriginally deposited solution FIG. 16 also shows how a misplaced drop ofsalt solution can differentially cover the array surface, which meansthat the signal between pairs of electrodes will be vastly differentdepending on their proximity to the salt crystal.

Therefore, in another embodiment of the systems and methods forcalibrating an osmolarity measuring device, a processing device can beconfigured to mathematically eliminate the effects of any residual saltcrystals from the osmolarity measurement of the sample at box 1614. Theeffects of the residual salt crystal can, for example, be eliminated byintegrating the descending curves from every electrode pair, whichestimates the amount of salt added to the solution. The estimation ofthe amount of salt that is added is accomplished by summing only thearea above the steady state line, which is determined by a linearregression far from the time point of sample delivery. These effects arethen subtracted out from the total volume of the sample. As previouslydiscussed, the total volume of the sample can be estimated by theprocessing device based on the number of electrodes that are in contactwith the sample.

Based on these parameters, the measured concentration of the sample isadjusted directly. The concentration of the sample is based on thenumber of ions per unit of volume. The osmolarity measurement providesthe total number of ions from the sample plus the residual salt crystal,and the processing device estimates the volume of the sample.Accordingly, the adjustment requires the subtraction of the numberresidual salt ions from the measured number of total ions in the sample.The number of residual salt ions is determined through the integrationmethod discussed above. This method enables the use of a salt solutionstandard on the microscale without the need for expensive washinghardware. After the effect from the residual salt has been subtracted,the processing device adjusts the resulting osmolarity measurement basedon the previously determined calibration factor at box 1514. The use ofthe calibration factor results in an osmolarity measurement that issubstantially independent from variances in the geometry of themicroelectrode array.

FIG. 19 is a flow chart that illustrates still another embodiment of amethod for calibrating an osmolarity measuring device with a standardfluid in accordance with the systems and methods described herein. Inthe embodiment of FIG. 19, a wash can be used in conjunction with theapplication of a standard fluid. The steps performed at boxes 1902,1904, and 1906 have been previously discussed and result in thedetermination of a calibration factor based on the measurement of one ormore standards. In one embodiment, the standard contains a simple saltsolution (NaCl), or a complex salt solution, with sodium, potassium,calcium and magnesium salts in physiological ratios).

At box 1908, an action is performed to remove the standard from the chipbefore the standard evaporates and prevent the accumulation of residualsalt on the chip. In one embodiment, the washing step uses amicrofluidic chamber attached in series to the sample receivingsubstrate to allow a steady stream of deionized water to flow across thechip surface. Once a standard aliquot has been deposited, either througha perpendicular microfluidic flow channel or by manual methods, and the(calibration measurement has been made, the washing apparatus will flowdeionized water across the electrode surface until the conductivity hasreached a steady state commensurate with the expected deionized waterlevels. The steady state conductivity indicates that the chip surfacehas been cleaned of any standard and is ready to accept a sample. Theflow is halted and the deionized water is allowed to evaporate on thechip surface, ideally leaving no salt crystal behind.

In another embodiment, a valved high pressure air supply can beimplemented to remove the standard. The tube is connected to the airsupply and placed in close proximity to the receiving substrate and atan acute angle from the surface. The angle is such that a quick puff ofair from the tube forces any fluid from the surface of the chip to becleared completely from the substrate. The flow of air is triggered uponcompletion of the calibration measurement. The resulting air flow may bepulsed several times until the signal at each electrode pair hasreturned to open circuit values. In another embodiment, air supply iscombined with the microfluidic washing stage to eliminate the need toevaporate fluids from the surface of the chip.

Furthermore, multipoint calibrations may be performed if a completewashing apparatus is attached to the chip surface, where deionized waterand increasingly concentrated salt solutions are deposited, or flowed,onto the chip surface, and then a puff of air is used to clear thearray. At boxes 1912 and 1914, the sample fluid is deposited onto themicro electrode array and the calibrated osmolarity measurement iscompleted in the methods that have been previously discussed.

Biomarker Normalization

In most patients who suffer from dry eye syndrome (DES), ocular allergy,general or ocular infections, blepharitis, diabetes, or other diseasesin which DNA or other molecular biomarkers are present in tears, thereis a clear clinical need for the ability to analyze nanoliter amounts oftears collected from the lower tear lake.

Nanoliter tear samples are necessary to minimize the time of residenceof a collection device within the tear lake, which lowers the chance ofinducing reflex tearing, a situation in which hypo-osmolar (lessconcentrated, very watery) tears are flushed onto the ocular surface,thereby reducing the available biomarker concentrations and introducingdiagnostic variability within the clinical routine. As the amount ofreflex tearing is disease-specific and patient-specific, the amount ofdilution varies with stimulation. Historically, tear collectionprotocols suggest collecting relatively large volumes of tears,typically several microliters of tears, in order to collect a sufficientsample volume to conduct standard in vitro diagnostic tests. Thesebiomarker assays often take upwards of thirty minutes of continual tearsampling to attain such high volumes. Older patients, and especiallythose with DES, often present less than 200 nL of available tears in thetear lake for sampling at a given time. Thus, tear collection forstandard in vitro tests is uncomfortable and moderately invasive.

Hypo-osmolar tears can result from a variety of conditions. Anoverabundance of non-lubricating tears can occur in certain dry eyesubtypes; known as epiphora, these patients may have occludednasolacrimal ducts which increase tear residence time within the tearlake.

Patients with DES are also known to have a dysfunction of the tear filmthat can result in a hyper-osmolar tear. Whether through aqueousdeficiency or meibornian gland disease, the steady state equilibriumconcentration of tears is significantly increased in DES patients. SomeDES patients are known to have steady state tear lake concentrationsapproximately 30%-50% higher than age-matched normals (healthysubjects). Measured osmolarities of 400 mOsm/L in the tear lake ofsevere DES patients have been frequently reported. Hyper-osmolar tearsare also observed in contact lens wearers. Regardless of contact lensmaterial or the type of lens worn, contact lenses disrupt the preoculartear film and shift the homeostasis of tears towards a hyper-osmolarstate.

Post-LASIK patients and DES patients may also have varying levels ofinnervation and/or nerve function, which affect the ability to producereflex tearing. In vitro diagnostics performed on these types ofpatients may therefore report quite different concentrations ofbiomarkers depending on the state of the patient and the manner in whichtears are collected. There is a clear need for in vitro diagnosticmethods that can eliminate the variability introduced by tear samplingand from hypo-osmolar and hyper-osmolar tear film concentrations.

Recently, a new class of microfluidic technologies have greatly reducedthe volume requirements for in vitro diagnostics, wherein submicrolitersamples can be used to test for biomarkers within tears. Because thetears offer an ideal, largely acellular biological matrix from which toperform various in vitro diagnostics, collecting tears may now be ofinterest to many doctors and medical professionals who are less familiarwith working near the ocular surface, and who may unknowingly causeundue reflex tearing during tear collection. The undue reflex tearingfrom such sampling can cause inaccurate diagnostic results. This problemreinforces the need for techniques of measuring biomarker concentrationsin tears that are independent of sampling.

In accordance with the present invention, biomarker normalization isperformed against a measured osmolarity in order to remove the impact oftear sampling and patient-specific tear homeostasis from theinterpretation of biomarker concentration in tears. The normalizationprovides an Adjusted Tear Biomarker Level.

Traditional measurement of tear biomarkers such as immunoglobulins (IgE,IgA, IgG, IgM), glucose, insulin levels, lactoferrin, tear lysozyme,cytokines, hormones, hormone metabolites, infectious disease phenotypes,nucleic acids, proteins, or lipid fractions, are carried out without thesimultaneous analysis of tear osmolarity. Traditional means of measuringtear osmolarity are incompatible with tear biomarker analysis. Inaccordance with the present invention, a receiving substrate, sampleregion, and energy transduction mechanism within a nanofluidic channelprovide for the first time, the possibility of measuring tear osmolarityon the same undiluted tear sample as the biomarker. The combination ofan integrated tear collection interface and transducer provides leverageagainst evaporation following sampling.

The calculation of an Adjusted Tear Biomarker Level is as follows.Normal tear osmolarity is generally accepted to be near 300 mOsm/L (withranges from 280-316 mOsm/L). The Adjusted Tear Biomarker Level can beobtained from the following equation:

Adjusted Tear Biomarker Level=(300 mOsm/L*Measured Tear BiomarkerLevel)/(Measured Tear Osmolarity Level)

The defined value of 300 mOsm/L can be substituted for any of theappropriate range of tear osmolarity levels. In another embodiment, thebasal level of tear osmolarity can be measured for a specific patient atthe beginning of a study, at pretreatment, at an early age, or prior tosurgery in order to establish a personalized baseline level of tearhomeostasis. Following the passage of time, a pharmaceuticaladministration, or surgery, the personalized baseline level can besubstituted for the defined 300 mOsm/L.

FIG. 20 is a flowchart that illustrates processing in accordance withthe normalization technique described herein. Initially, at the boxnumbered 2002, an aliquot volume (such as a nanoliter tear volume) ofsample fluid is collected to a sample region of a sample chip. Next, atbox 2004, an osmolarity output signal is received from the sample regionthat indicates energy properties of the sample fluid, wherein theosmolarity output signal is correlated with osmolarity of the samplefluid. Next, at box 2006, a biomarker output signal is received from thesample region that indicates chemical properties of the sample fluid,wherein the biomarker output signal is correlated with biomarkerconcentration of the sample fluid. Next, at box 2008, the osmolarityoutput signal is processed to produce an osmolarity value for the samplefluid and the biomarker output signal is processed to produce abiomarker concentration value for the sample fluid. The processing canbe performed simultaneously or serially. Lastly, at box 2010, theAdjusted Biomarker Level is determined, which provides normalization ofbiomarker concentration values. As noted above, the adjusted levelprovides a normalization of biomarker concentration values and cancorrect for patient-specific tear homeostasis and clinician induced tearsampling variance in connection with obtaining the sample fluid.

The operations depicted in FIG. 20 can be performed by any of the systemembodiments illustrated in the drawings (FIGS. 1-11) with a processorconfigured to perform the normalization operations as described herein.

If desired, open loop adjustment is also possible, where the 300 mOsm/Lconstant is unused, as in the following equation:

Open Loop Adjusted Tear Biomarker Level=Measured Tear BiomarkerLevel/(Measured Tear Osmolarity Level)

An advantage of using a standard or personal baseline osmolarity valueto normalize against is that the Adjusted Tear Biomarker Level isexpressed in units identical to the Measured Tear Biomarker Level. Openloop adjustment would result in a Biomarker Level/mOsms/L, which couldbe a more difficult parameter for clinicians to interpret, especially ifthe analyte of interest is commonly known to have a range inunnormalized units.

Similar Adjusted Tear Biomarker Levels can be performed using linear,logarithmic, exponential, or through the use of calibration curveadjustments. For example, a linear adjusted Tear Biomarker Level couldtake on the form given by:

Linear Adjusted Tear Biomarker Level=B _(adj) =B_(m)*(1+(Alpha*(Osm_(m)−300 mOsms/L)))

where B_(adj) is the Linear Adjusted Tear Biomarker Level, B_(m) is themeasured biomarker level, Alpha is the linear correction factor, andOsm_(m) is the measured osmolarity. Both the Alpha and the 300 mOsms/Lpoint can be altered to fit the specific biomarker curve.

IgE, for example, is suggested to be found on the order of 50-60 ofng/mL range in unsensitized individuals, and 100-300 ng/mL in patientswith vernal, seasonal, or perennial conjunctivitis (see publications byNomura, “Tear IgE Concentrations in Allergic Conjunctivitis” in Eye,Vol. 12 (Part 2), 1998 at 296-98; and Allansmith, “Tissue, Tear, andSerum IgE Concentrations in Vernal Conjunctivitis” in Am. J. ofOphthalmology, Vol 81, No. 4, 1976, at 506-11). From Nomura:

-   -   Tear IgE concentrations showed significant increases in the        vernal keratoconjunctivitis (322.2+/−45.7 ng/ml), seasonal        allergic conjunctivitis (194.7+/−21.7 ng/ml) and perennial        allergic conjunctivitis (134.8+/−23.1 ng/ml) groups when        compared with controls (52.1+/−9.7 ng/ml, p<0.01). No        significant difference was found between epidemic        keratoconjunctivitis (97.2+/−11.7 ng/ml) and bacterial        conjunctivitis (92.6+/−13.8 ng/ml) groups and controls (p=0.1).

For DES patients with an elevated osmolarity of 400 mOsm/L, unnormalizeddetermination of the tear IgE levels may easily lead to incorrectinterpretation. A more severe bacterial conjunctivitis could easily bemistaken for a relatively mild perennial allergic conjunctivitis basedon unnormalized IgE. Similarly, if a normal patient with seasonalallergic conjunctivitis was overstimulated during tear collection andproduced hypo-osmolar reflex tears, their tear IgE levels could easilydrop beneath perennial allergic conjunctivitis indications. Normalizingby measured tear osmolarity prevents this type of misdiagnosis.

In one embodiment, a plurality of electrodes contained within the sampleregion of the receiving substrate can be functionalized with distinctenergy transduction mechanisms; one set of electrodes would contain anosmolarity transducer (e.g., a non-polarizing metal electrode forimpedance analysis of osmolarity such as gold, platinum, and the like,and a conductive polymer such as polypyrrole, polyacetylene,polyaniline, and the like) with another set of electrodes configured asan electrochemical transducer for a specific biomarker (e.g., a sandwichor competitive assay comprising a bare metal or conductive polymercoated electrode with corresponding surface chemistry to bind antibody,avibody, aptamer, or other receptor for a the biomarker ligand). In thisembodiment, the osmolarity is multiplexed in space. An example of thisembodiment is shown in FIG. 21, which is a plan view of the receivingsubstrate 2100 showing the sample region 2102. A detail view of theelectrodes 2104 in the sample region 2102 is provided in FIG. 22. Theillustrated electrodes 2104 indicate a group of electrodes demarcatedwithin the sample region as group “A” with the biomarker function andgold osmolarity electrodes demarcated within the sample region as group“B” for the osmolarity function. A capillary 2106 receives the samplefluid and distributes the fluid along its length for interaction withthe electrodes A and B.

Upon depositing an aliquot volume of sample fluid on the sample regionof a substrate (through capillary action, aspiration, or similartechniques), energy imparted into the sample fluid is transduced by thesample region to produce an output signal that indicates the energyproperties of the sample fluid that are correlated with the osmolarityof the sample fluid. Simultaneously or in parallel operations,potentiometric, amperometric, pulse voltammetry, cyclic voltammetry,broadband frequency response, impedance, or other electrochemicalmethods are used to transduce output signals from the electrochemicallymodified electrodes to indicate chemical properties of the sample fluidthat are correlated with the concentration of biomarkers in tears. Thus,the osmolarity and biomarker output signals are generated at the sametime but from different sets of electrodes. Subsequently, an AdjustedTear Biomarker Level is calculated to compensate for the possibility ofpatient hyperosmolarity or dilution introduced by tear sampling. Thatis, the Adjusted Tear Biomarker Level can compensate and correct forpatient-specific tear homeostasis and for clinician-induced tearsampling variance in connection with obtaining the sample fluid.

In other embodiments where osmolarity is multiplexed in space, opticalindicators, such as a plurality of nano-scale spheres having aluminescence correlated to osmolarity of the sample fluid are depositedon a subset of the sample region. Other optical transduction mechanismscan include iontophoretic fluorescent nanoscale spheres, or metal filmsamenable to surface plasmon resonance. In parallel, subsets of thesample region are configured to produce output signals that indicatechemical properties of the sample fluid that are correlated with theconcentration of a biomarker in tear. Sample region subsets can includeluminescence, fluorescent, chemiluminescent, resonant energy transfer,optoentropic, surface enhanced Raman, colorimetric, surface plasmonresonant, plasmonic, or other optical indicators commonly used forbiomarker transduction. Following illumination by an optical energysource that imparts optical energy into the sample fluid, the opticalenergy can be transduced by the sample region to produce an opticaloutput signal that indicates the energy and chemical properties of thesample fluid that are correlated with the osmolarity and tear biomarkerconcentration, respectively. An optical detector then receives theoptical output signal from the sample region, and a processing deviceprocesses the output signal to produce an estimate of sample fluidosmolarity and biomarker concentration. Subsequently, an Adjusted TearBiomarker Level is calculated to compensate for the possibility ofpatient hyperosmolarity or dilution introduced by tear sampling.

In yet another embodiment, electrical, optical, or thermal (e.g.,freezing point depression) methods of osmolarity determination withinthe receiving substrate can be independently combined with electrical oroptical methods of tear biomarker concentration detection. For example,conductive osmolarity determinations can be combined with opticaltransducers for tear biomarker analysis. Spatial multiplexing supportsmultiple biomarkers in such a format.

In spatial multiplexing embodiments, the measurement of tear osmolaritycan either be performed at the same time as the biomarker assays, orserially by modulating the input energy type.

For example, if both osmolarity and tear biomarker analysis arespatially multiplexed via optical methods, then tear osmolarity can bedetermined by surface plasmon resonance (i.e., the angle atop a metalfilm) and the tear biomarker can be analyzed by fluorescence.

In another embodiment, electrodes covered with a chromogenic competitiveassay system can be interrogated for conductivity in order to determineosmolarity, followed by absorbance of light in order to quantify theconcentration of tear biomarker.

If fluorescent nanoscale spheres are used as an osmolarity marker andchemiluminescent reporter antibodies are used to transduce the tearbiomarker concentration, then the first input would comprise anappropriate excitation light, and the second energy input would comprisepumping a known concentration of luminescent substrate and fuel acrossthe sample region (e.g., luminol and hydrogen peroxide).

In another embodiment, a “molecular ruler” could be used to transducethe osmolarity, for example, a plasmonic pair of nanoscale metal spheresattached to DNA could indicate the bulk sample fluid osmolarity by theoptical detection of absorbance change around 520 nm. In parallel, iffluorescently labeled secondary antibodies are used to label the analyteof interest, the fluorescent response from excitation light would beread following the absorbance of the molecular ruler within the samefluid.

Other combinations of electrical, optical, and thermal transduction canbe combined to achieve requisite levels of sensitivity, specificity, andmultiplexing while minimizing the need for washing or externalinterfacing to the sample region.

These methods are generally amenable to spatial multiplexing in adiscrete sense, where subsets of the sample region are orthogonal withinthe surface plane. Such methods are also amenable to vertical spatialmultiplexing, where, for example, the biomarker transducer is built atopthe osmolarity transducer, as in a fluorescent assay built atop aconductive polymer.

In another embodiment, a plurality of electrodes are configured forelectrochemical transduction of the biomarker of interest, and theosmolarity is multiplexed in time. In this embodiment, all theelectrodes are functionalized with the same surface chemistry for thebiomarker assay. Because there is a diffusion time associated with theligand binding of the tear biomarker, osmolarity can be determinedimmediately after introduction into the sample region, prior to theelectrodes being substantially affected by the presence of analyte. Inone embodiment, electrochemical assays where the Debye layer ismodulated by the tear biomarker assay and is detected by a change iscapacitance of the system, the baseline reading can be correlated totear osmolarity, and the dynamic change in capacitance over time canindicate the levels of tear biomarker. Thus, the osmolarity andbiomarker output signals are produced from the same electrodes but areseparated in time, the osmolarity output occurring substantiallyimmediately upon introduction of the sample fluid to the sample regionand the biomarker output occurring following the requisite diffusiontime for the sample region.

Other embodiments allow for the osmolarity to be determined at adifferent frequency spectrum than the biomarker assay. For example, theosmolarity can be determined by a 10-100 kHz impedance spectra, and thetear biomarker concentration analyzed by a DC or low frequencyamperometric or voltammetric steady state measurement. Alternatively,the osmolarity can be determined by a 10-100 kHz impedance spectra, andthe tear biomarker concentration analyzed by a 100 kHz-GHz excitednanostructure spectra, or THz adsorption spectra. Other combinations ofpulsed, or sinusoidal electrochemical measurements, including theaddition of a small sinusoidal signal atop a square wave input, can beused for such analyses.

Other aspects in accordance with the invention can include analysis oftear osmolarity and tear biomarkers to be analyzed in parallelnanofluidic chambers, and then normalized against each other.

Still other aspects of the invention include for the implementationwhere two separate tear samples are taken and analyzed in serial. Serialanalyses of tear biomarker and tear osmolarity would give an indirectestimate the impact of sampling. It is likely that sequential analysis,if performed properly, would give a better indication of the tearhomeostasis than unnormalized biomarker analysis alone.

While certain embodiments have been described above, it will beunderstood that the embodiments described are by way of example only.Accordingly, the inventions should not be limited based on the describedembodiments. Rather, the scope of the inventions described herein shouldonly be limited in light of the claims that follow when taken inconjunction with the above description and accompanying drawings.

1-37. (canceled)
 38. A fluid measuring system for measuring osmolarityof a sample fluid and concentration of a biomarker within the samplefluid, said system comprising: a) a sample chip comprising a samplefluid; an optical energy source that illuminates the sample fluid; andan optical detector that receives optical energy from the illuminatedsample fluid in response to imparting energy into the sample fluid, andwhich processes (i) optical energy properties of the sample fluid toprepare an osmolarity output signal which corresponds to osmolarity ofthe sample fluid, and (ii) chemical properties of the sample fluid toprepare a biomarker output signal which corresponds to concentration ofa biomarker within the sample fluid; and b) a processing device that:receives the osmolarity output signal from the sample fluid andprocesses the osmolarity output signal to produce an osmolarity valuefor the sample fluid; and receives the biomarker output signal from thesample fluid and processes the biomarker output signal to produce abiomarker concentration value for the sample fluid.
 39. The fluidmeasuring system of claim 38, wherein the biomarker is IgE, IgA, IgG,IgM, glucose, insulin, lactoferrin, a cytokine, a hormone, a hormonemetabolite, an infectious disease phenotype, a nucleic acid, a protein,or a lipid fraction.
 40. The fluid measuring system of claim 38, furthercomprising normalization of biomarker concentration values.
 41. Thefluid measuring system of claim 40, wherein the normalization ofbiomarker concentration values corrects for patient-specific tearhomeostasis.
 42. The fluid measuring system of claim 40, wherein thenormalization of biomarker concentration values corrects for clinicianinduced tear sampling variance in connection with obtaining the samplefluid.
 43. The fluid measuring system of claim 38, wherein the analysesof osmolarity and of the biomarkers are performed simultaneously. 44.The fluid measuring system of claim 38, wherein the analyses ofosmolarity and of the biomarkers are performed serially.
 45. The fluidmeasuring system of claim 40, wherein the normalization of biomarkerconcentration values is linear.
 46. The fluid measuring system of claim40, wherein the normalization of biomarker concentration values isratiometric.
 47. The fluid measuring system of claim 40, wherein thenormalization of biomarker concentration values is exponential.
 48. Thefluid measuring system of claim 40, wherein the normalization ofbiomarker concentration values is based on a calibration curve.
 49. Asystem as defined in claim 38, wherein the optical detector includes aphotodiode, wherein the optical detector includes a photodiode.